In this study, we investigated the effects of membrane cholesterol content on the mechanical properties of cell membranes by using optical tweezers. We pulled membrane tethers from human embryonic kidney cells using single and multi-speed protocols, and obtained time-resolved tether forces. We quantified various mechanical characteristics including the tether equilibrium force, bending modulus, effective membrane viscosity, and plasma membrane–cytoskeleton adhesion energy, and correlated them to the membrane cholesterol level. Decreases in cholesterol concentration were associated with increases in the tether equilibrium force, tether stiffness, and adhesion energy. Tether diameter and effective viscosity increased with increasing cholesterol levels. Disruption of cytoskeletal F-actin significantly changed the tether diameters in both non-cholesterol and cholesterol-manipulated cells, while the effective membrane viscosity was unaffected by F-actin disruption. The findings are relevant to inner ear function where cochlear amplification is altered by changes in the membrane cholesterol content.